1% BSA. Cells were then permeabilized and blocked in a solution of 0. 1 M PBS with 1% BSA, 5% skim milk and 0. 3% Triton X 100 for 60 min at room temperature. Cells were incubated in blocking solution containing poly clonal antibodies raised against the carb oxyl and sellckchem amino terminals of the and B GM CSF recep tor subunits, respectively. Inhibitors,Modulators,Libraries After three washes with PBS, cells were incubated with anti rabbit, anti goat and anti mouse IgGs conjugated to Alexa Fluor 488 and 594 nm, respectively. Cells were again washed three times in PBS and mounted under coverslips in a solution containing 4, 6 diamidino 2 phenylindole. Samples were examined under confocal microscope and photos were obtained using photomicroscopy.

In vitro maturation of cumulus oocyte complexes After follicular aspiration, COC were classified into five groups based on the morphology of their surrounding Inhibitors,Modulators,Libraries cumulus cells. Group A with many layers of com pact cumulus cells. Group B with partially removed cumulus cells. Group C denuded oocytes. Group D degeneration of oocyte cytoplasm. Group E expanded cumulus cells. Only COC classified as Group A and B were used in this study. Cumulus oocyte complexes were then washed twice in PBS Dulbecco and twice in maturation media according to each treat ment. Maturation media consisted of SOF at pH 7. 4 supplemented with aminoacids BME 50. MEM 100x, BSA FV and gentamicin. Cumulus oocyte complexes were randomly assigned to SOF medium supplemented with Recombinant human GM CSF at con centrations of 1, 10 and 100 ng ml.

Two additional groups were incorporated in the experimental design SOF alone and a positive control maturation media Inhibitors,Modulators,Libraries consisted of tissue culture medium supplemented with 10% FBS, 0. 2 uM Pyruvate, 5 ug ml LH, 40 mg ml FSH and 50 ug ml gentamicin. Groups of 10 15 COC were allocated for in vitro ma turation in 50 ul droplets of treatment media in Petri dishes under mineral oil for 22 h in humidified at mosphere consisting in 5% CO2 at 38. 5 C. Assessment of cumulus expansion and oocyte nuclear maturation After 22 h of IVM, oocytes were collected and evaluated according to the cumulus expansion and then nuclear maturation. Cumulus expansion was determined using three different methods 1 higher and a lower diameter for each COC were measured using Inhibitors,Modulators,Libraries a micrometric rule previously calibrated using a 0. 1 mm objective.

2 oocytes were microphotographed and higher and lower diameters were measured using a Fluoview software. and 3 a subjective scale was used to estimate the degree of cumulus expansion. The degree of cumulus expansion was measured as follows 0, no expansion. 1, separation of only the outermost layer of Inhibitors,Modulators,Libraries cumulus cells. 2, further expansion involving the outer half of the cumulus oophorus. selleck chem 3, further expansion up to, but not including, the corona radiate. 4, complete expansion, including the innermost corona radiate cells.